Camps M, Hou C, Sidiropoulos D, Stock J B, Jakobs K H, Gierschik P
Pharmakologisches Institut, Universität Heidelberg, Federal Republic of Germany.
Eur J Biochem. 1992 Jun 15;206(3):821-31. doi: 10.1111/j.1432-1033.1992.tb16990.x.
We have previously shown that soluble fractions obtained from human HL-60 granulocytes contain a phospholipase C which is markedly stimulated by the stable GTP analogue guanosine 5'-[3-O-thio]triphosphate (Camps, M., Hou, C., Jakobs, K. H. and Gierschik, P. (1990) Biochem. J. 271, 743-748]. To investigate whether this stimulation was due to a soluble alpha subunit of a heterotrimeric guanine-nucleotide-binding protein or a soluble low-molecular-mass GTP-binding protein, we have examined the effect of purified guanine-nucleotide-binding protein beta gamma dimers on the phospholipase-C-mediated formation of inositol phosphates by HL-60 cytosol. We found that beta gamma subunits, purified from bovine retinal transducin (beta gamma t), markedly stimulated the hydrolysis of phosphatidylinositol 4,5-bisphosphate by this phospholipase C preparation. The stimulation of phospholipase C by beta gamma t was not secondary to a phospholipase-A2-mediated generation of arachidonic acid, was prevented by the GDP-liganded transducin alpha subunit and was additive to activation of phospholipase C by guanosine 5'-[3-O-thio]triphosphate. Beta gamma t also stimulated soluble phospholipase C from human and bovine peripheral neutrophils, as well as membrane-bound, detergent-solubilized phospholipase C from HL-60 cells. Stimulation of soluble HL-60 phospholipase C was not restricted to beta gamma t, but was also observed with highly purified beta gamma subunits from bovine brain. Fractionation of HL-60 cytosol by anion-exchange chromatography revealed the existence of at least two distinct forms of phospholipase C in HL-60 granulocytes. Only one of these forms was sensitive to stimulation by beta gamma t, demonstrating that stimulation of phospholipase C by beta gamma subunits is isozyme specific. Taken together, our results suggest that guanine-nucleotide-binding protein beta gamma subunits may play an important and active role in mediating the stimulation of phospholipase C by heterotrimeric guanine-nucleotide-binding proteins.
我们之前已经表明，从人HL-60粒细胞获得的可溶性组分含有一种磷脂酶C，它受到稳定的GTP类似物鸟苷5'-[3-O-硫代]三磷酸的显著刺激（坎普斯，M.，侯，C.，雅各布斯，K.H.和吉尔斯基克，P.（1990年）《生物化学杂志》271，743 - 748）。为了研究这种刺激是由于异源三聚体鸟嘌呤核苷酸结合蛋白的可溶性α亚基还是可溶性低分子量GTP结合蛋白引起的，我们研究了纯化的鸟嘌呤核苷酸结合蛋白βγ二聚体对HL-60细胞溶质中磷脂酶C介导的肌醇磷酸形成的影响。我们发现，从牛视网膜转导素（βγt）纯化的βγ亚基显著刺激了这种磷脂酶C制剂对磷脂酰肌醇4,5-二磷酸的水解。βγt对磷脂酶C的刺激不是继发于磷脂酶A2介导的花生四烯酸生成，被GDP结合的转导素α亚基所抑制，并且与鸟苷5'-[3-O-硫代]三磷酸对磷脂酶C的激活具有加和性。βγt还刺激了人和牛外周中性粒细胞中的可溶性磷脂酶C，以及HL-60细胞膜结合的、去污剂增溶的磷脂酶C。对可溶性HL-60磷脂酶C的刺激不仅限于βγt，从牛脑高度纯化的βγ亚基也能观察到这种刺激。通过阴离子交换色谱对HL-60细胞溶质进行分级分离揭示了HL-60粒细胞中至少存在两种不同形式的磷脂酶C。这些形式中只有一种对βγt的刺激敏感，表明βγ亚基对磷脂酶C的刺激具有同工酶特异性。综上所述，我们的结果表明鸟嘌呤核苷酸结合蛋白βγ亚基可能在介导异源三聚体鸟嘌呤核苷酸结合蛋白对磷脂酶C的刺激中发挥重要且积极的作用。
